Caveolae and lipid rafts are related low density, detergent-resistant membrane domains. They are thought to be involved in the process of signal transduction since a variety of signaling components including the EGF receptor, have been shown to be highly enriched in these domains. We have shown that intact lipid rafts are required for proper function of EGF-stimulated PI turnover and that cholesterol modulates EGF receptor binding and kinase activities. These findings suggest that lipid rafts are important contributors to the regulation of EGF receptor-mediated signaling. To understand the role of lipid rafts in EGF receptor signaling, four specific aims are proposed: 1) Analyze the distribution of EGF receptors between lipid rafts and the non-raft compartment in intact cells. 2) Identify the domains(s) of the EGF receptor that are responsible for targeting the receptor to lipid rafts. 3) Compare the enzymatic properties of EGF receptors in raft and on-raft membrane fractions. And, 4) Evaluate the mechanism through which caveolin-1 induces inhibition of EGF receptor kinase activity. Two non-invasive, fluorescence-based methods, fluorescence photobleaching recovery and fluorescence correlation spectroscopy, will be used to determine the relative proportion of rapidly diffusing (non-raft) and slowly diffusing (raft) GFP-EGF receptors in intact cells. Domain swapping between the EGF receptor and the VSV-G protein, a non-raft protein, will be used to identify portions of the EGF receptor responsible for directing the receptor into lipid rafts. The effect of raft localization and cholesterol levels on the function of the EGF receptor will be measured directly by assessing receptor binding and kinase activity in raft and non-raft membrane fractions in vitro. The mechanism through which the caveolar protein, caveolin- 1 inhibits EGF receptor kinase activity will be investigated by mutagenesis of the proposed sites of interaction in these two proteins and by analysis of the potential role played by cholesterol in caveolin-1 -mediated inhibition of the EGF receptor kinase. Together, these studies will determine how and to what extent the EGF receptor partitions into lipid rafts. They will also delineate the effect of raft localization on EGF receptor function. These findings will provide insight into the contribution of lipid rafts to the regulation of EGF receptor-mediated signaling.